CN111630059B - Novel radiometal-binding compounds for diagnosis or treatment of cancers expressing prostate-specific membrane antigen - Google Patents

Abstract

The present application relates to compounds of formula I-a or formula I-b, or salts or solvates thereof. ^{R 1} is ‑(CH ₂ ) ₅ CH ₃ or contains 2‑4 fused benzene rings. R ² is I, Br, F, Cl, H, OH, OCH ₃ , NH ₂ , NO ₂ or CH ₃ . R ³ is a peptide-bound glycine, aspartic acid or glutamate, or a glutamic acid peptide bound by C _delta . L is ‑CH ₂ NH‑, ‑(CH ₂ ) ₂ NH‑, ‑(CH ₂ ) ₃ NH‑ or ‑(CH ₂ ) ₄ NH‑. ^{R 4} is a radioactive metal chelator, optionally bound to a radioactive metal. The variable “n” is 1‑3. The compound can be used to image tissue expressing prostate-specific membrane antigen (PSMA), or to treat diseases expressing PSMA (such as cancer).

Description

Novel radiometal binding compounds for diagnosing or treating cancers expressing prostate specific membrane antigen

Technical Field

The present invention relates to radiolabeled compounds, particularly prostate specific membrane antigen targeting compounds, for use in the selective imaging or treatment of cancer.

Background

Prostate Specific Membrane Antigen (PSMA) is a transmembrane protein that catalyzes the hydrolysis of N-acetyl-aspartyl glutamate to glutamate and N-acetyl aspartate. ¹ PSMA is not expressed in most normal tissues, but is overexpressed (up to 1000-fold) in prostate tumors and metastases. ^2-3 Based on their pathological expression patterns, a variety of radiolabelled PSMA-targeting constructs were designed and evaluated for use in the internal radiation treatment of prostate cancer. ^4-7

Common radiolabeled PSMA-targeted inward radiotherapy formulations are derivatives of lysine-urea-glutamate (Lys-urea-Glu), including ¹³¹I-MIP-1095、¹⁷⁷ Lu-PSMA-617 and ¹⁷⁷Lu-PSMA I&T.^5-7, of which ¹⁷⁷ Lu-PSMA-617 is the most studied drug, currently being evaluated in multicenter assays. ^7-14 Preliminary data indicate that ¹⁷⁷ Lu-PSMA-617 is effective in treating metastatic prostate cancer, with 32-60% of patients having PSA levels reduced by more than 50% and without serious side effects. ^7-13 In phase II studies conducted in australia, objective responses were observed in 82% of patients with measurable lymph node or visceral disease. ¹⁴ But the complete remission rate was low (< 7%) and up to 33% of patients still had progressive disease after ¹⁷⁷ Lu-PSMA-617 treatment. ^7,9-13 Interestingly, recent reports indicate that ²²⁵ Ac-PSMA-617 (replacing ¹⁷⁷ Lu with α -emitter ²²⁵ Ac) has an impressive therapeutic effect in patients with advanced metastatic prostate cancer, including a subject whose disease has progressed after ¹⁷⁷ Lu-PSMA-617 treatment. ¹⁵

Although ²²⁵ Ac-PSMA-617 has great potential for internal radiotherapy, the supply of ²²⁵ Ac is limited worldwide. The more potent ¹⁷⁷ Lu-labeled PSMA-targeting formulation has a greater direct impact on the internal radiation treatment of prostate cancer than ²²⁵ Ac-PSMA-617, since ¹⁷⁷ Lu, which is in Good Manufacturing Practice (GMP), is available in large numbers from multiple suppliers. ²²⁵ The greater efficacy of Ac-PSMA-617 may be due to the highly linear energy transfer of the alpha-particles, resulting in double strand breaks that make them less susceptible to radiation resistance than indirect damage caused by beta-particles emitted by ¹⁷⁷ Lu. One way to increase the effectiveness of radiation therapy is to increase the radiation dose deposited in the tumor per unit of administered radioactive ¹⁷⁷ Lu-labeled agent. Improving delivery of ¹⁷⁷ Lu to tumors can also reduce the cost of therapeutic radiopharmaceuticals by reducing radioisotope costs.

Any information presented above is not intended or construed as prior art to the present invention.

Disclosure of Invention

Disclosed herein are novel PSMA-targeting compounds.

The present disclosure provides compounds of formula I-a or formula I-b, or a salt or solvate of formula I-a or formula I-b:

Wherein:

R ¹ is Or- (CH ₂)₅CH₃;

R ² is I, br, F, cl, H, OH, OCH ₃,NH₂,NO₂ or CH ₃;

r ³ is

L is-CH ₂NH-,-(CH₂)₂NH-,-(CH₂)₃ NH-, or- (CH ₂)₄ NH-;

R ⁴ is a radioactive metal chelator, optionally bound to a radioactive metal X, and n is 1-3.

Also disclosed are compounds having formula II or salts or solvates of formula II:

Wherein R ² is I, br or methyl, n is 1-3;X absent, ²²⁵ Ac or ¹⁷⁷ Lu.

In some embodiments, when X is a diagnostic radiometal (e.g., suitable for imaging but not necessarily limited to ⁶⁴Cu、¹¹¹In、⁸⁹Zr、⁴⁴Sc、⁶⁸Ga、^99mTc、⁸⁶Y、¹⁵² Tb or ¹⁵⁵ Tb), these compounds can be used to image PSMA expressing cancers in a subject. Accordingly, a method of imaging a PSMA-expressing cancer in a subject is also disclosed, the method comprising administering to the subject a composition comprising the compound and a pharmaceutically acceptable excipient, and imaging tissue of the subject.

In some embodiments, when X is a therapeutic radiometal (e.g., a toxic radiometal, but not limited to ⁶⁴Cu、⁶⁷Cu、⁹⁰Y、¹¹¹In、^{114 Minute (min)} 、^117mSn、¹⁵³Sm、¹⁴⁹Tb、¹⁶¹Tb、¹⁷⁷Lu、²²⁵Ac、²¹³Bi、²²⁴Ra、²¹²Bi、²¹²Pb、²²⁵Ac、²²⁷Th、²²³Ra、⁴⁷Sc、¹⁸⁶Re or ¹⁸⁸ Re), such compounds may be used to treat PSMA-expressing cancers in a subject. Thus, also disclosed are methods of treating a cancer that expresses Prostate Specific Membrane Antigen (PSMA) in a subject, comprising administering to the subject a composition comprising the compound and a pharmaceutically acceptable excipient.

This summary may not describe all features of the invention.

Drawings

To describe the above-mentioned and other features of the present invention in detail, the following detailed description will be made by reference to the accompanying drawings:

FIG. 1 shows a representative shift curve of ¹⁸ F-DCFPyL for binding to LNCaP prostate cancer cells by assays performed in triplicate by Lu-PSMA-617 and Lu-HTK 01169.

FIG. 2 shows SPECT/CT images of (A) ¹⁷⁷ Lu-labeled PSMA-617 and (B) HTK01169 in LNCaP tumor-bearing mice. Higher and sustained uptake of ¹⁷⁷ Lu-HTK01169 was observed in tumor xenografts.

FIG. 3A shows the biodistribution of ¹⁷⁷ Lu-PSMA-617 selected organs in mice bearing LNCaP tumor burden (n≥5). The strips are arranged from left to right for 1 hour, 4 hours, 24 hours, 72 hours and 120 hours.

FIG. 3B shows the biodistribution of ¹⁷⁷ Lu-HTK01169 in selected organs in mice bearing LNCaP tumor burden (n≥5). The strips are arranged from left to right for 1 hour, 4 hours, 24 hours, 72 hours and 120 hours.

FIG. 4 shows the radiation dose (mGy/MBq) delivered to the major organs/tissues of 25g mice by ¹⁷⁷ Lu-HTK01169 (left column) and ¹⁷⁷ Lu-PSMA-617 (right column) calculated using OLINDA software.

FIG. 5 shows the radiation dose (mGy/MBq) of ¹⁷⁷ Lu-PSMA-617 (bottom) and ¹⁷⁷ Lu-HTK01169 (top) on LNCaP tumor-loaded mice, calculated using OLINDA software. These data were obtained with different tumors, but assuming that ¹⁷⁷ Lu-PSMA-617 and ¹⁷⁷ Lu-HTK01169 had the same tumor uptake (% ID, percent injected dose) and residence time.

FIG. 6 shows a line graph of the overall survival of LNCaP tumor-loaded mice (8 per group) injected with saline (control group), ¹⁷⁷ Lu-PSMA-617 (18.5 MBq) or ¹⁷⁷ Lu-HTK01169 (2.3-18.5 MBq). Median survival from shortest to longest control ,2.3MBq ¹⁷⁷Lu-HTK01169,18.5MBq ¹⁷⁷Lu-PSMA-617,4.6MBq ¹⁷⁷Lu-HTK01169,9.3MBq ¹⁷⁷Lu-HTK01169 and 18.5MBq ¹⁷⁷ Lu-HTK01169.

Figure 7 shows a line graph of (a) tumor volume and (B) body weight over time after treatment of mice with physiological saline.

FIG. 8 shows a plot of (A) tumor volume and (B) body weight over time after treatment of mice with ¹⁷⁷ Lu-PSMA-617 (18.5 MBq).

FIG. 9 shows a plot of (A) tumor volume and (B) body weight over time after treatment of mice with ¹⁷⁷ Lu-HTK 01169 (18.5 MBq).

FIG. 10 shows a plot of (A) tumor volume and (B) body weight over time after treatment of mice with ¹⁷⁷ Lu-HTK 01169 (9.3 MBq).

FIG. 11 shows a plot of (A) tumor volume and (B) body weight over time after treatment of mice with ¹⁷⁷ Lu-HTK 01169 (4.6 MBq).

FIG. 12 shows a plot of (A) tumor volume and (B) body weight over time after treatment of mice with ¹⁷⁷ Lu-HTK 01169 (2.3 MBq).

Figure 13 shows PET/CT images of maximum intensity projections obtained 1 hour or 3 hours after injection of ⁶⁸Ga-HTK03026、⁶⁸Ga-HTK03027、⁶⁸ Ga-HTK03029 and ⁶⁸ Ga-HTK03041 in mice bearing LNCaP tumor burden. All ⁶⁸ Ga-labeled compounds are excreted mainly via the renal route. ⁶⁸Ga-HTK03026、⁶⁸ Tumor uptake of Ga-HTK03027 and ⁶⁸ Ga-HTK03029 was comparable, while ⁶⁸ Ga-HTK03041 had the highest tumor uptake, which increased from 1 hour to 3 hours after injection.

Figure 14 shows PET/CT images of maximum intensity projections obtained 1 hour and 3 hours after injection of ⁶⁸Ga-HTK03055、⁶⁸ Ga-HTK03056 and ⁶⁸ Ga-HTK03058 in mice bearing LNCaP tumor burden. All three compounds showed some degree of blood retention as the heart was clearly visible in the image 1h after injection. Although the uptake in blood (heart) decreases with time (1 to 3 hours after injection), the uptake in tumors increases with time.

Figure 15 shows PET/CT images of maximum intensity projections obtained 1 hour and 3 hours after injection of ⁶⁸Ga-HTK03082、⁶⁸ Ga-HTK03085 and ⁶⁸ Ga-HTK03086 in mice bearing LNCaP tumor burden. All three compounds are mainly excreted by the renal route. ⁶⁸ Ga-HTK03085 and ⁶⁸ Ga-HTK03086 show significantly higher blood retention than ⁶⁸ Ga-HTK 03082. Tumor uptake of ⁶⁸ Ga-HTK03085 and ⁶⁸ Ga-HTK03086 also increased over time, 1 to 3 hours after injection.

Figure 16 shows PET/CT images of maximum intensity projections obtained 1 hour and 3 hours after injection of ⁶⁸Ga-HTK03087、⁶⁸ Ga-HTK03089 and ⁶⁸ Ga-HTK03090 in mice bearing LNCaP tumor burden. ⁶⁸ Ga-HTK03089 and ⁶⁸ Ga-HTK03090 show significantly higher blood retention than ⁶⁸ Ga-HTK 03087. Tumor uptake of ⁶⁸ Ga-HTK03089 and ⁶⁸ Ga-HTK03090 also increased over time, 1 to 3 hours after injection.

Detailed Description

The terms "comprising," "having," "including," and "containing," and their grammatical variants, as used herein, are inclusive or open-ended and do not exclude additional, unrecited elements and/or method steps. When used herein in connection with a composition, use, or method, the term "consisting essentially of means that other elements and/or method steps may be present, but that such additions do not materially affect the recited composition, method, or functional manner of use. The term "consisting of" when used herein in connection with a composition, use, or method means that no other elements and/or method steps are present.

Compositions, uses, or methods described herein as comprising certain elements and/or steps may in certain embodiments also be comprised primarily of such elements and/or steps, and in other embodiments such elements and/or steps, whether or not such embodiments are specifically mentioned. The use or method described herein as comprising certain elements and/or steps may in certain embodiments also be comprised primarily of such elements and/or steps, in other embodiments whether or not such embodiments are specifically mentioned.

The indefinite article "a" or "an" does not exclude the possibility that a plurality of elements is present, unless the context clearly requires that there be only one element. The singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Herein, "a" or "an" when used in conjunction with "comprising" means "one", but it may also mean "one or more", "at least one", and "one or more than one".

Unless otherwise indicated, "certain embodiments," "various embodiments," "one embodiment," and similar terms include a particular feature described for that embodiment alone or in combination with any other embodiment or embodiments described herein, whether or not other embodiments are directly or indirectly referenced, and whether or not the feature or embodiment is described in the context of methods, products, uses, compositions, compounds, and the like.

As used herein, the terms "treat," "therapeutic," and the like include improving symptoms, reducing disease progression, improving prognosis, and reducing cancer recurrence.

The term "diagnostic reagent" as used herein includes "imaging reagent". Thus, "diagnostic radiometals" include radiometals suitable for use as imaging agents.

The term "subject" refers to an animal (e.g., a mammal or a non-mammal). The subject may be a human or a non-human primate. The subject may be a laboratory mammal (e.g., mouse, rat, rabbit, hamster, etc.). The subject may be an agricultural animal (e.g., horse, sheep, cow, pig, camel, etc.) or a livestock (e.g., canine, feline, etc.).

As used herein, the terms "salt" and "solvate" have the usual meaning in chemistry. Thus, when the compound is a salt or solvate, it is combined with a suitable counterion. How to prepare salts or exchange counterions is well known in the art. Typically, these salts can be prepared by reacting the free acid forms of these compounds with a stoichiometrically appropriate base (e.g., including, but not limited to, na, ca, mg or K hydroxides, carbonates, bicarbonates, etc.), or by reacting the free base forms of these compounds with a stoichiometrically appropriate acid. Such reactions are generally carried out in water or in an organic solvent or in a mixture of both. The counter ion may be changed by, for example, ion exchange techniques such as ion exchange chromatography. All zwitterionic, salts, solvates and counterions are in the generic form, unless a specific form is specifically indicated.

In certain embodiments, the salt or counterion can be pharmaceutically acceptable for administration to a subject. More generally, with respect to any of the pharmaceutical compositions disclosed herein, non-limiting examples of suitable excipients include any suitable buffering agents, stabilizers, salts, antioxidants, complexing agents, tonicity agents, cryoprotectants, lyoprotectants, suspending agents, emulsifiers, antibacterial agents, preservatives, chelating agents, binders, surfactants, wetting agents, non-aqueous carriers such as fixed oils, or polymers for sustained or controlled release. See, for example, berge et al 1977 (journal of pharmaceutical science 66:1-19), or Lemington-pharmaceutical science and practice, 21 st edition (Gennaro et al, edit. Lippincott Williams & WILKINS PHILADELPHIA), each of which is incorporated herein by reference in its entirety.

In one aspect of the invention, compounds of formula I-a or formula I-b, or salts or solvates of formula I-a or formula I-b are disclosed:

Wherein:

R ¹ is Or- (CH ₂)₅CH₃;

R ² is I, br, F, cl, H, OH, OCH ₃,NH₂,NO₂ or CH ₃;

r ³ is

L is-CH ₂NH-,-(CH₂)₂NH-,-(CH₂)₃ NH-, or- (CH ₂)₄ NH-;

R ⁴ is a radioactive metal chelator, optionally bound to a radioactive metal X, and n is 1-3.

Wave lineThe symbols shown by the bonds in the formulae (e.g., formula I-a or formula I-b) are intended to define the R groups (e.g., R ¹、R² and R ³) on one side of the wavy line without changing the structural definition on the opposite side of the wavy line. When the R groups are bonded at two or more lateral ends (e.g., R ³), atoms outside the wavy line are included to elucidate the R groups. Thus, only the atoms between the two wavy lines constitute the R group.

In some embodiments, the compound is of formula I-a or a salt or solvate of formula I-a.

In some embodiments, the compound is of formula I-b or a salt or solvate of formula I-b.

In some embodiments, R ¹ isIn some embodiments, R ¹ is

In some embodiments, R ¹ isIn some embodiments, R ¹ isIn some embodiments, R ¹ is- (CH ₂)₅CH₃).

R ¹ forms a side chain of an amino acid residue (e.g., 2-naphthylalanine, etc.). In some embodiments, the amino acid is an L-amino acid, i.e(E.g., L-2-naphthylalanine, etc.). In some embodiments, the amino acid is a D-amino acid(E.g., D-2-naphthylalanine, etc.).

In some embodiments, R ¹ isIn some embodiments, R ¹ is

In some embodiments, R ¹ isIn some embodiments, R ¹ is

In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3.

As shown in the formulas I-a and I-b, only one R ² group is arranged on the benzene ring. When not hydrogen, R ² may be para, meta, or ortho on the benzene ring, i.e.:

Or alternatively Or alternatively

In some embodiments, R ² is in the para-position. In some embodiments, R ² is in the meta-position. In some embodiments, R ² is in the ortho position.

In some embodiments, R ² is H. In some embodiments, R ² is I. In some embodiments, R ² is Br. In some embodiments, R ² is F. In some embodiments, R ² is Cl. In some embodiments, R ² is OH. In some embodiments, R ² is OCH ₃. In some embodiments, R ² is NH ₂. In some embodiments, R ² is NO ₂. In some embodiments, R ² is CH ₃.

In some embodiments, R ³ is(I.e., gly residues).

In some embodiments, R ³ is(I.e., asp residues). In some embodiments, the Asp residue is D-Asp. In some embodiments, asp is L-Asp.

In some embodiments, R ³ is(I.e., glu residues). In some embodiments, the Glu residue is D-Glu. In some embodiments, the Glu residue is L-Glu.

In some embodiments, R ³ is

In some embodiments, R ³ is

In some embodiments, R ³ is

R ⁴ can be any radiometal chelator that can bind to the target radiometal (i.e., X) and which is functionalized to attach to an amino group. Many suitable radiometal chelators are known, for example as summarized in Price and Orvig, chem. Soc. Rev.,2014,43,260-290, which is incorporated by reference in its entirety. In some embodiments, R ⁴ is:

DOTA (1, 4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid) or derivatives thereof, including, for example, but not limited to, dotga;

TETA (1, 4,8, 11-tetraazacyclotetradecane-1, 4,8, 11-tetraacetic acid) or derivatives thereof, including, for example, but not limited to CB-TE2A (4, 11-bis- (carboxymethyl) -1,4,8, 11-tetraazabicyclo [6.6.2] -hexadecane);

SarAR (1-N- (4-aminobenzyl) -3,6,10,13,16,19-hexaazabicyclo [6.6.6] -eicosane-1, 8-diamine) or derivatives thereof;

NOTA (1, 4, 7-triazacyclononane-1, 4, 7-triacetic acid) or derivatives thereof, including, for example, but not limited to, nodga;

TRAP (1, 4, 7-triazacyclononane-1, 4, 7-trimethyl (2-carboxyethyl) phosphinic acid) or a derivative thereof;

HBED (N, N0-bis (2-hydroxybenzyl) -ethylenediamine-N, N0-diacetic acid) or a derivative thereof;

2,3-HOPO (3-hydroxypyridin-2-one) or a derivative thereof;

PCTA (3,6,9,15-tetraazabicyclo [9.3.1] -pentadec-1 (15), 11, 13-triene-3, 6,9, -triacetic acid) or derivatives thereof;

DFO (deferoxamine) or derivatives thereof, for example including but not limited to tetrahydrooximate DFO-star;

DTPA (diethylenetriamine pentaacetic acid) or derivatives thereof, including for example, but not limited to CHX-DTPA (2- (p-benzyl isothiocyanate) -cyclohexyldiethylenetriamine pentaacetic acid);

OCTAPA (N, N0-bis (6-carboxy-2-pyridylmethyl) -ethylenediamine-N, N0-diacetic acid) or a derivative thereof (e.g., picolinic acid derivative), or

H2-MACROPA (N, N' -bis [ (6-carboxy-2-pyridinyl) methyl ] -4, 13-diaza-18-crown-6) or a derivative thereof.

In some embodiments, X is absent.

In some embodiments, X is a therapeutic radiometal. For example, but not limited to, X may be ⁶⁴Cu、⁶⁷Cu、⁹⁰Y、¹¹¹In、^{114 Minute (min)} 、^117mSn、¹⁵³Sm、¹⁴⁹Tb、¹⁶¹Tb、¹⁷⁷Lu、²²⁵Ac、²¹³Bi、²²⁴Ra、²¹²Bi、²¹²Pb、²²⁵Ac、²²⁷Th、²²³Ra、⁴⁷Sc、¹⁸⁶Re or ¹⁸⁸ Re. In some embodiments, X is ⁶⁴ Cu. In some embodiments, X is ⁶⁷ Cu. in some embodiments, X is ⁹⁰ Y. In some embodiments, X is ¹¹¹ In. In some embodiments, X is 114 minutes. In some embodiments, X is ^117m Sn. In some embodiments, X is ¹⁵³ Sm. In some embodiments, X is ¹⁴⁹ Tb. In some embodiments, X is ¹⁶¹ Tb. In some embodiments, X is ¹⁷⁷ Lu. In some embodiments, X is ²²⁵ Ac. In some embodiments, X is ²¹³ Bi. In some embodiments, X is ²²⁴ Ra. In some embodiments, X is ²¹² Bi. In some embodiments, X is ²¹² Pb. In some embodiments, X is ²²⁵ Ac. In some embodiments, X is ^227Th. In some embodiments, X is ²²³ Ra. In some embodiments, X is ⁴⁷ Sc. In some embodiments, X is ¹⁸⁶ Re. In some embodiments, X is ¹⁸⁸ Re.

In some embodiments, X is a diagnostic radiometal. For example, but not limited to, X may be ⁶⁴Cu、¹¹¹In、⁸⁹Zr、⁴⁴Sc、⁶⁸Ga、^99mTc、⁸⁶Y、¹⁵² Tb or ¹⁵⁵ Tb. In some embodiments, X is ⁶⁴ Cu. In some embodiments, X is ¹¹¹ In. In some embodiments, X is ⁸⁹ Zr. In some embodiments, X is ⁴⁴ Sc. In some embodiments, X is ⁶⁸ Ga. In some embodiments, X is ^99m Tc. In some embodiments, X is ⁸⁶ Y. In some embodiments, X is ¹⁵² Tb. In some embodiments, X is ¹⁵⁵ Tb.

In some embodiments, R ¹ isR ³ isWherein R ² is I, br, F, cl, H, OH, OCH ₃,NH₂,NO₂, or CH ₃, and wherein X, ⁹⁰Y、⁶⁷Ga、⁶⁸Ga、¹⁷⁷Lu、²²⁵ Ac, or ¹¹¹ In is absent. In certain embodiments, R ² is in the para-position. In certain embodiments, R ² is 1. In certain embodiments, X is ¹⁷⁷ Lu, while in other embodiments, X is ²²⁵ Ac.

In some embodiments, R ¹ isR ³ isWherein R ² is I, br, F, cl, H, OH, OCH ₃,NH₂,NO₂, or CH ₃, and wherein X, ⁹⁰Y、⁶⁷Ga、⁶⁸Ga、¹⁷⁷Lu、²²⁵ Ac, or ¹¹¹ In is absent. In certain embodiments, R ² is in the para-position. In certain embodiments, R ² is l. In certain embodiments, X is ¹⁷⁷ Lu, while in other embodiments, X is ²²⁵ Ac. In certain embodiments, n is 3.

In some embodiments, L is-CH 2NH-. In some embodiments, L is- (CH ₂)₂ NH-) in some embodiments, L is- (CH ₂)₃ NH-. In some embodiments, L is- (CH ₂)₄ NH-.

L forms a side chain of an amino acid residue (e.g., 2, 3-diaminopropionic acid (Dap), 2, 4-diaminobutyric acid (Dab), ornithine (Orn), or lysine (Lys)). In some embodiments, the amino acid is an L-amino acid, i.e(E.g., L-Dap, L-Dab, L-Orn or L-Lys). In some embodiments, the amino acid is a D-amino acid(E.g., D-Dap, D-Dab, D-Orn or D-Lys).

In some embodiments, the amino acid residue formed from L is an L-amino acid, and the amino acid residue formed from R ¹ is also an L-amino acid. In some embodiments, the amino acid residue formed by L is a D-amino acid and the amino acid residue formed by R ¹ is also a D-amino acid. In some embodiments, the amino acid residue formed from L is an L-amino acid and the amino acid residue formed from R ¹ is a D-amino acid. In some embodiments, the amino acid residue formed from L is a D-amino acid and the amino acid residue formed from R ¹ is an L-amino acid.

In some embodiments, the compound has formula II or is a salt or solvate of formula II:

Wherein R ² is I, br or methyl, n is 1-3;X absent, ²²⁵ Ac or ¹⁷⁷ Lu. In some embodiments, R ² is I. In some embodiments, R ² is Br. In some embodiments, R ² is methyl. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, X is absent. In some embodiments, X is ¹⁷⁷ Lu and is incorporated into the DOTA group. In some embodiments, X is ²²⁵ Ac and is incorporated in the DOTA group.

In some embodiments, the compound has formula III or is a salt or solvate of formula III:

Wherein X is absent, or ⁹⁰Y、⁶⁷Ga、⁶⁸Ga、¹⁷⁷Lu、²²⁵ Ac or ¹¹¹ In. When X _in is ¹⁷⁷ Lu, the compound has the following structure, or a salt or solvate thereof:

The synthetic schemes for HTK01169 and Lu-HTK01169 are provided in example 1 below. Example 2 provides a synthetic scheme for preparing various metal chelating PSMA binding compounds that incorporate various options for the R groups of formulas I-a and I-b.

The compounds modulate albumin binding and PSMA binding (as compared to Lu-PSMA-617) to modulate (e.g., enhance) tumor uptake/retention, thereby providing alternative or improved diagnostic or therapeutic agents for PSMA-expressing cancers. In particular, the above compounds comprise an albumin binding domain, i.e(E.g., iodophenylbutyryl in Lu HTK 01169; see also PCT patent publication No. WO 2008/053360), which increases the blood circulation time of the compound. Modification of the albumin binding group by changing the value of R ² and/or n (i.e., n=1, 2, or 3) and/or introducing R ³ (e.g., adding Gly or carboxylate-containing Asp or Glu) can modulate (increase or decrease) the albumin binding strength (i.e., binding affinity) of the compound, thereby modulating the final blood circulation time of the compound. Without wishing to be bound by theory, compounds that bind too strongly to albumin (i.e. bind too with affinity to albumin) will stay in the blood circulation for a long time and accumulation in the tumor will be very low. This will result in a decrease in the total uptake of the tumor and an excessive dose of radiation to the bone marrow. Meanwhile, if the binding affinity of albumin is too weak, the compound will clear too quickly from the blood circulation, reducing the chance of accumulation in the tumor. In addition, the above compounds also contain a Lys-ureido-Glu PSMA binding moiety. The PSMA binding strength of a compound may be modulated (increased or decreased) by modifying R ¹. Without wishing to be bound by theory, the adjustable tumor uptake/retention capacity of the above compounds may be due to modulation of albumin binding and/or PSMA binding strength (as compared to Lu-PSMA-617). Diagnostic or therapeutic efficacy can be further modulated by altering the chelator and the bound radiometal. As shown in the examples below, the variables described above were adjusted in the above compounds to enhance PSMA-expressing tumor uptake/retention and thus enhance diagnosis or efficacy.

When X is a diagnostic radiometal, certain embodiments of the compounds are disclosed for use in the preparation of a radiolabeled tracer for imaging PSMA-expressing tissue in a subject. Also disclosed is a method of imaging PSMA expressing tissue in a subject, wherein the method comprises administering to the subject a composition comprising certain dosing regimens of the compound and a pharmaceutically acceptable excipient, and imaging the tissue of the subject, e.g., using positron emission computed tomography (PET). When the tissue is diseased (e.g., PSMA-expressing cancer), PSMA-targeted therapies may be selected to treat the subject.

When X is a therapeutic radiometal, the use of certain embodiments of the compound (or pharmaceutical composition thereof) for treating a disease expressing PSMA (e.g., cancer) in a subject is disclosed. Thus, there is provided the use of the compound in the manufacture of a medicament for the treatment of a PSMA-expressing disease in a subject. Also provided is a method of treating a PSMA-expressing disease in a subject, wherein the method comprises administering to the subject a composition comprising the compound and a pharmaceutically acceptable excipient. For example, but not limited to, the disease may be PSMA-expressing cancer.

PSMA expression has been detected in a variety of cancers (e.g., rowe et al, 2015, journal of nuclear medicine 29:877-882; satheskig et al, 2015, journal of European nuclear medicine and molecular imaging 42:1482-1483; verburg et al, 2015, journal of European nuclear medicine and molecular imaging 42:1622-1623; and Pyka et al, journal of nuclear medicine 2015, 11, 19, jnumed. 115.164442). Thus, without limitation, the PSMA-expressing cancer may be prostate cancer, kidney cancer, breast cancer, thyroid cancer, gastric cancer, colorectal cancer, bladder cancer, pancreatic cancer, lung cancer, liver cancer, brain tumor, melanoma, neuroendocrine tumor, ovarian cancer, or sarcoma. In some embodiments, the cancer is prostate cancer.

The invention will be further illustrated in the following examples.

EXAMPLE 1 ¹⁷⁷ Lu-HTK01169

1.1 Materials and methods

1.11 General procedure

All chemicals and solvents were commercial products and were used without further purification. Human serum for protein binding analysis was obtained from Innovative Research (novo, michigan). PSMA-617 and HTK01169 were synthesized by solid phase methods on a Aapptec (path Yi Siwei mol, kentucky) Endeavor 90 peptide synthesizer. Mass analysis was performed using an AB SCIEX (framingham, ma) 4000QTRAP mass spectrometer system with ESI ion source. Purification and quality control of non-radioactive and ¹⁷⁷ Lu-labeled peptides was performed on an agilent (santa clara, california) HPLC system equipped with a type 1200 quaternary pump and a type 1200 UV absorbance detector. The radioactive HPLC system was equipped with a Bioscan (Washington, D.C.) sodium iodide scintillation detector. The HPLC columns used were Phenominex (Tuna C18, 5. Mu., 250X 10 mm) semi-preparative column (Luna C18, 5. Mu., 250X 4.6 mm) and Phenominex analytical column (Luna C18, 5. Mu., 250X 4.6 mm). ¹⁷⁷ Radioactivity of Lu-tagged peptides was performed using Capintec (lambda, new jersey)The dose calibrator performs the measurements.

1.12 Solid phase Synthesis of PSMA-617 and HTK01169

The synthesis of PSMA-617 and its albumin binder derivative HTK01169, starting from Fmoc-Lys (ivDde) -Wang resin, has been improved according to the reported procedure. ¹⁶ After coupling the isocyanate of the tert-butyl protected glutamyl moiety, ¹⁷ was used to remove the ivDde protecting group with 2% hydrazine in N, N-Dimethylformamide (DMF). Fmoc-2-Nal-OH, fmoc-tranexamic acid and DOTA-tris (t-bu) ester were then coupled, followed by cleavage with trifluoroacetic acid (TFA) to give the crude product of PSMA-617. PSMA-617 was obtained in 25% yield after HPLC purification with 25% acetonitrile containing 0.1% tfa using a semi-preparative chromatography column at a flow rate of 4.5 mL/min (t _R =10.5 min). ESI-MS, PSMA-617C ₄₉H₇₂N₉O₁₆ calculated [ M+H ] ⁺ 1042.5, found [ M+H ] ⁺ 1042.6.

To synthesize HTK01169, fmoc-Lys (ivDde) -OH was coupled to the Fmoc-tranexamic acid post sequence. Extension was continued by adding Fmoc-Glu (tBu) -OH and 4- (p-iodophenyl) butanoic acid at the N-terminus. Subsequently, the ivDde protecting group was removed with 2% hydrazine in DMF and DOTA-tris (t-bu) ester was coupled to Lys side chain. The peptide was cleaved by TFA treatment and purified by HPLC using a semi-preparative column with 37% acetonitrile in water containing 0.1% TFA at a flow rate of 4.5 mL/min (t _R =9.7 min). The yield of HTK01169 was 21%. ESI-MS HTK01169C ₇₀H₁₀₀N₁₂O₂₁ I calculated [ M+H ] ⁺ 1571.6, found [ M+H ] ⁺ 1571.7.

1.13 Synthesis of Lu-PSMA-617 and Lu-HTK01169

A solution of PSMA-617 (5.5 mg, 5.3. Mu. Mol) or HTK01169 (4.1 mg, 2.6. Mu. Mol) was incubated with LuCl ₃ (5 eq) in NaOAc buffer (0.1M, 500. Mu.L, pH 4.2) for 15 min at 90℃and then purified by HPLC using a semi-preparative column. For Lu-PSMA-617, hplc conditions were 25% acetonitrile in water, 0.1% tfa, flow rate of 4.5 mL/min (tr=9.7 min). The yield was 62%. ESI-MS calculated for Lu-PSMA-617C ₄₉H₆₉N₉O₁₆ [ Lu ] [ M+H ] ⁺ 1214.4, found [ M+H ] ⁺ 1214.4. For Lu-HTK01169, HPLC conditions were 37% acetonitrile in water, 0.1% tfa, flow rate was 4.5 mL/min (t _R =10.0 min). The yield was 31%. ESI-MS calculated for Lu-HTK 01169C ₇₀H₉₇N₁₂O₂₁ I [ Lu ] [ M+H ] ⁺ 1743.5, found [ M+H ] ⁺ 1743.9.

1.14 In vitro competitive binding assay

In vitro competition binding assays were performed using LNCaP prostate cancer cells and ¹⁸ F-DCFPyL as radioligands as described previously. ¹⁸ Briefly, LNCaP cells (400,000 cells/well) were seeded onto 24-well poly-D-lysine coated plates for 48 hours. The growth medium was removed and replaced with HEPES buffered saline (50mM HEPES,pH 7.5,0.9% sodium chloride) and the cells were incubated at 37 ℃ for 1 hour. ¹⁸ F-DCFPyL (0.1 nM) was added to each well (in triplicate) containing various concentrations (0.5 mM-0.05 nM) of test compound (Lu-PSMA-617 or Lu-HTK 01169). Nonspecific binding was determined in the presence of 10 μm of nonradiolabeled DCFPyL. The assay mixture was further incubated for 1 hour at 37 ℃ with gentle agitation. The buffer and hot ligand were then removed and the cells were washed twice with cold HEPES buffered saline. To harvest the cells, 400 μl of 0.25% trypsin solution was added to each well. Radioactivity was measured on a Wizard2 2480 automatic gamma counter from PerkinElmer (waltherm, ma). Nonlinear regression analysis and K _i calculation were performed using GRAPHPAD PRISM software.

1.15 Synthesis of ¹⁷⁷ Lu-PSMA-617 and ¹⁷⁷ Lu-HTK01169

¹⁷⁷LuCl₃ (329.3-769.9 MBq, 10-20. Mu.L) was added to a solution of PSMA-617 or HTK01169 (25. Mu.g) in NaOAc buffer (0.5mL,0.1M,pH 4.5). The mixture was incubated at 90 ℃ for 15 minutes, then purified by HPLC. ¹⁷⁷ HPLC purification conditions (semi-preparative column, 4.5 mL/min) for Lu-PSMA-617 and ¹⁷⁷ Lu-HTK01169 were 23% and 36% acetonitrile in water (0.1% TFA), respectively. ¹⁷⁷ The residence times of Lu-PSMA-617 and ¹⁷⁷ Lu-HTK01169 were 15.0 minutes and 13.8 minutes, respectively. Quality control was performed on an analytical column with a flow rate of2 mL/min using the corresponding purification solvent conditions. ¹⁷⁷ The residence time of both Lu-PSMA-617 and ¹⁷⁷ Lu-HTK01169 was around 5.5 minutes.

1.16 Plasma protein binding assay

Plasma protein binding assays were performed according to literature methods. ¹⁹ Briefly, 37KBq of ¹⁷⁷ Lu-PSMA-617 or ¹⁷⁷ Lu-HTK 01169 in 50. Mu.L of PBS was added to 200. Mu.L of human serum and the mixture was incubated for 1 min at room temperature. The mixture was then loaded onto a membrane filter (Nanosep, 30K,Pall Corporation,USA) and centrifuged for 45 minutes (30,130×g). Normal saline (50 μl) was added and centrifugation was continued for 15 min. The top with membrane filter and the bottom with solution were counted on a gamma counter. The control group replaced human serum with physiological saline.

1.17 SPECT/CT imaging, biodistribution and internal radiation therapy studies

SPECT/CT imaging and biodistribution were performed using NOD-scid IL2Rgamma ^null (NSG) male mice, and internal radiation therapy studies were performed using NOD.Cg-Rag1 ^tmlMom Il2rg^tm1wjl/SzJ (NRG) male mice. Mice were housed and tested according to guidelines established by the Canadian animal protection Committee and approved by the animal ethics Committee of the university of Combria, dobex. Mice were anesthetized by inhalation of 2% isoflurane in oxygen and 1×10 ⁷ LNCaP cells were implanted subcutaneously behind the left shoulder. Mice were used for the study when tumors reached 5-8mm in diameter 5-6 weeks after inoculation.

SPECT/CT imaging experiments were performed using MILabs (Utrehler, netherlands) U-SPECT-II/CT scanner. Each tumor-bearing mouse was injected under anesthesia with about 37MBq of ¹⁷⁷ Lu-labeled PSMA-617 or HTK 01169 (2% isoflurane in oxygen) via the tail vein. At 4, 24, 72 and 120 hours post injection, mice were allowed to resume consciousness and freely walk in cages and image. At each time point, mice were again sedated and placed in the scanner. A CT scan was first performed for 5 minutes, as an anatomical reference, with a voltage set at 60kV and a current set at 615 ua, and then a static emission scan of 60 minutes mice was acquired in list mode using an ultra-high resolution multi-pinhole (1 mm pinhole size) collimator. The data were reconstructed using U-SPECT II software with 20% window width over three energy windows. The photo peak window is centered at 208keV, and the low and high scattering windows are centered at 170 and 255keV, respectively. Images were reconstructed using an ordered subset expectation maximization algorithm (3 iterations, 16 subsets) and a 0.5mm post-processing gaussian filter. Image decay was corrected to injection time at PMOD (PMOD Technologies, switzerland) followed by conversion to DICOM for qualitative visualization in Inveon Research Workplace software (SIEMENS MEDICAL Solutions USA, inc.).

For biodistribution studies, mice were injected with ¹⁷⁷ Lu-labeled PSMA-617 or HTK01169 (2-4 MBq) as described above. At predetermined time points (1, 4, 24, 72 or 120 hours after injection), mice were euthanized by inhalation of CO ₂. Blood is immediately drawn from the heart and the target organ/tissue is collected. The collected organs/tissues were weighed and counted using an automatic gamma counter. For the blocking study, mice were co-injected with ¹⁷⁷ Lu-HTK01169 (2-4 MBq) and 50nmol of nonradioactive standard solution and the target organs/tissues were collected 4h after injection.

For radiation therapy studies, tumor-bearing mice were injected with normal saline (control group), ¹⁷⁷ Lu-PSMA-617 (18.5 MBq) or ¹⁷⁷ Lu-HTK01169 (18.5, 9.3, 4.6 or 2.3 MBq) (n=8 per group). Tumor size and body weight were measured twice weekly from the day of injection (day 0) to the end of the study (day 120). Endpoint criteria were defined as weight loss >20%, tumor volume >1000mm ³, or tumor active ulcers.

1.18 Radiation dose calculation

Internal dose estimates were calculated using organ level internal dose assessment (OLINDA) software v.2.0. ³⁷. These estimates were calculated using a 25g MOBY phantom for mice, a NURBS model for adult males, ³⁹ a previously reported unit density region model for tumors. ⁴⁰ All body models and region models were available in olinoda and required input of total attenuation number normalized by injection activity in mbq×h/MBq for each source organ/tumor.

Biodistribution data (see tables 1 and 2 below) were used to determine the kinetic input values required by OLINDA. First, each value decays to its corresponding time point (the values in the table are shown at the time of injection). Then, using internal software developed by Python, different time points (% ID/g) of each organ uptake data were fitted to a single indexAnd double indexA function. The best fit is selected based on the determined coefficients (R2) that maximize the fit and minimizing the residual. The area under the curve is calculated analytically based on the parameters obtained from the best fit for each organ, which provides the kinetic input value required by OLINDA.

In the case of mice, adrenal glands, blood, fat, muscle and seminal vesicles were not modeled in the model. These organs are grouped together and contained in the rest of the body as described by OLINDA.

The biodistribution data of mice were extrapolated to humans using the method proposed by Kirschner et al ⁴¹, as shown in the following formula:

Where M _Organ is the mass of the organ and M represents the total mass of the body. The subscripts indicate whether these values correspond to humans or mice. Organ mass and total weight were taken from human body mass simulated in olinoda. Since the biodistribution data did not distinguish between left colon, right colon and rectum in the olinoda manikin, it was assumed that these three regions of the intestine had the same active uptake (% ID/g) biodistribution as the large intestine. Let the% ID/g of blood be the% ID/g of the heart content of the model. This value was also used to calculate bone marrow uptake according to the method described by Wessels et al ⁴², and we assumed a hematocrit of 0.40 based on the patient values shown by this study. Finally, the red marrow value used a blood measurement of 0.32 times. In the case of humans, fat, muscle and seminal vesicles in the biodistribution data are not modeled in the model, so the number of decays in these areas is contained in the rest of the body. The data for the mouse case were again fitted and the total number of decays in MBq×h/MBq was input to OLINDA.

Finally, the number of decays in the tumor was also calculated based on the biodistribution data of the mice and the values were input into the available region model in OLINDA.

1.2 Results

1.21 Peptide Synthesis and radiochemistry

PSMA-617 and HTK01169 were synthesized in 25% and 21% yields, respectively. After reaction with LuCl ₃, purification by HPLC gave Lu-PSMA-617 and Lu-HTK01169 in 62% and 31% yields, respectively. The MS verifies the identity of PSMA-617, HTK01169 and its LU ligands.

¹⁷⁷ Lu labeling was performed in 90℃acetate buffer (pH 4.5) followed by HPLC purification. The resulting ¹⁷⁷ Lu-PSMA-617 had a radiochemical yield of 86.0±1.7% (n=3), a molar activity of 782±43.3 GBq/. Mu.mol and a radiochemical purity of >99%. The resulting ¹⁷⁷ Lu-HTK01169 had a radiochemical yield of 63.0±16.2% (n=4), a molar activity of 170±73.6 GBq/. Mu.mol and a radiochemical purity of >99%.

1.22 Binding to PSMA and serum proteins

Lu-PSMA-617 and Lu-HTK01169 inhibited ¹⁸ F-DCFPyL binding to PSMA on LNCaP cells in a dose-dependent manner (fig. 1), and they calculated K _i values of 0.24±0.06 and 0.04±0.01nM (n=3), respectively. After incubation with saline and centrifugation, the filter layers of ¹⁷⁷ Lu-PSMA-617 and ¹⁷⁷ Lu-HTK01169 had combined radioactivity of 5.21±1.42 and 25.8±3.42% (n=3), respectively. Under the same conditions, the filtration-bound radioactivity of ¹⁷⁷ Lu-PSMA-617 and ¹⁷⁷ Lu-HTK01169 was increased to 82.7±0.32 and 99.2±0.02% (n=3), respectively, with human serum instead of physiological saline.

1.23SPECT/CT imaging and biodistribution

SPECT/CT imaging studies showed that ¹⁷⁷ Lu-PSMA-617 and ¹⁷⁷ Lu-HTK01169 were excreted mainly by the renal route, especially at early time points (4 and 24 hours, fig. 2), with higher renal retention of ¹⁷⁷ Lu-HTK 01169. Higher and sustained tumor uptake was observed in ¹⁷⁷ Lu-HTK 01169. ¹⁷⁷ Biodistribution data for Lu-PSMA-617 and ¹⁷⁷ Lu-HTK01169 are shown in FIGS. 3A and 3B (see also tables 1 and 2). These data are consistent with the observations of SPECT/CT images.

TABLE 1 biological distribution data of ¹⁷⁷ Lu-PSMA-617 in mice with LNCaP xenografts.

TABLE 2 biological distribution data of ¹⁷⁷ Lu-HTK01169 in mice with LNCaP xenografts.

¹⁷⁷ Lu-PSMA-617 is rapidly cleared from blood and non-target organs/tissues. Only 0.68.+ -. 0.23% ID/g remained in the blood 1 hour after injection. Uptake was observed in tissues expressing PSMA, including spleen (3.34.+ -. 1.77% ID/g), adrenal gland (4.88.+ -. 2.41% ID/g), kidney (97.2.+ -. 19.4% ID/g), lung (1.34.+ -. 0.39% ID/g) and LNCaP tumor (15.1.+ -. 5.58% ID/g). ^20-21 Tumor uptake gradually decreased to 7.91±2.82% id/g 120 hours after injection. The contrast of ¹⁷⁷ Lu-PSMA-617 tumor to background increased over time due to faster clearance from other tissues/organs (see table 1 above).

Using the built-in albumin binder, ¹⁷⁷ Lu-HTK01169 had a relatively lower blood clearance than ¹⁷⁷ Lu-PSMA-617 (FIGS. 3A and 3B). ¹⁷⁷ Tumor uptake of Lu-HTK01169 was continuously increased at the early time point, peaked 24 hours after injection (55.9±12.5% id/g), and continued during the study (56.4±13.2% id/g for 120 hours). Similar to ¹⁷⁷ Lu-PSMA-617, uptake was also observed in spleen, adrenal glands, kidneys and lungs (Table 2 above). ¹⁷⁷ The contrast of the tumor to background of Lu-PSMA-617 also increases over time due to the continuous uptake of the tumor and relatively rapid clearance from other organs/tissues. Blocking with cold standards reduced uptake in all tissues/organs collected, particularly PSMA expressing kidneys (125±16.4% versus 5.50±1.95% id/g) and LNCaP tumors (55.9±12.5% versus 1.70±0.28% id/g), compared to biodistribution data collected at the same time point (4 h).

1.24 Radiation dose calculation

Based on biodistribution data obtained from tumor bearing mice, an estimate of the radiation dose delivered to the major organs/tissues of the mice was calculated using OLINDA software. The results are shown in fig. 4 and table 3, where source organ input kinetics (MBq-h/MBq) and target organ dose (mGy/MBq) calculated from the data fitting are shown. Compared to ¹⁷⁷ Lu-PSMA-617, ¹⁷⁷ Lu-HTK01169 provided 9.4 to 23.1 times the high dose to all major organs except the bladder, which received 1.5 times the high dose from ¹⁷⁷ Lu-PSMA-617.

TABLE 3 radiation dose (mGy/GBq) of 25g mice major organs calculated using OLINDA software.

Similar results were obtained for the calculated radiation dose delivered to the human organ/tissue (table 4). Most human organs/tissues will obtain a 11.9 to 24.9 times higher radiation dose from ¹⁷⁷ Lu-HTK 01169. Notably, using ⁷⁷ Lu-HTK01169, the brain, heart, red bone marrow, and spleen will receive 6.0-fold, 50.4-fold, 30.4-fold, and 28.1-fold high doses. The bladder will receive a dose of ¹⁷⁷ Lu-PSMA-617 1.3 times higher.

TABLE 4 radiation dose (mGy/GBq) of major human organs (men) calculated using OLINDA software.

The radiation dose delivered to the unit density region according to the LNCaP tumor kinetics of ¹⁷⁷ Lu-PSMA-617 and ¹⁷⁷ Lu-HTK01169 is shown in FIG. 5 and Table 5. ¹⁷⁷ The kinetic absorption values of Lu-PSMA-617 and ¹⁷⁷ Lu-HTK01169 were 3.80MBq-h/MBq and 31.72MBq-h/MBq, respectively, as input values to OLINDA. The dose of ¹⁷⁷ Lu-HTK01169 to LNCaP tumors was 8.3 times higher than ¹⁷⁷ Lu-PSMA-617, regardless of the size of the simulated region (tumor).

TABLE 5 radiation dose (mGy/MBq) calculated from the unit density area model of LNCaP tumors.

1.25 Internal radiation therapy study

The results of the internal radiation treatment study are shown in Table 6 and FIG. 6, and the LNCaP tumor volume and the change in mouse body weight over time after treatment are shown in FIGS. 7-12. The tumor volume of the control group (group a in table 6, fig. 7 (a)) continued to increase after treatment (saline injection), and the median survival period of the control group was only 14 days (euthanasia of mice when their tumor volume reached 1000mm ³). Tumors in mice treated with ¹⁷⁷ Lu-PSMA-617 (18.5 MBq, panel B in table 6, fig. 8A) initially shrink, but later resume growth, resulting in an increase in median survival to 58 days. The tumor size change over time in mice treated with ¹⁷⁷ Lu-HTK01169 (group C-F in table 6, fig. 9 (a) -12 (a)) was dependent on injections with higher radioactivity, resulting in more effective and longer tumor growth inhibition. Median survival for mice groups treated with ¹⁷⁷ Lu-HTK01169 at 18.5, 9.3, 4.6 and 2.3MBq was greater than 120, 103, 61 and 28 days, respectively. No weight loss was observed for all mice regardless of treatment (fig. 7 (B) -12 (B)), and all mice treated with ¹⁷⁷ Lu-HTK01169 of 18.5MBq survived to the end of the study (day 120).

Table 6 data from radiotherapy studies including median survival after treatment of tumors with physiological saline, ¹⁷⁷ Lu-PSMA-617 or ¹⁷⁷ Lu-HTK 01169.

1.3 Discussion

The use of small molecule albumin binders to extend the circulation time of the drug and maximize its tumor uptake has become an attractive strategy for designing internal radiation therapeutics. This pioneering work was mainly done by the scientist of the Federal regulatory institute of Zurich, who used D-Lys acylated on the epsilon-amino group with 4- (p-iodophenyl) butanoic acid as the albumin binding motif. ²² Previous studies have focused on applying this strategy to the design of folate receptor targeted radiopharmaceuticals. ²³ Radiolabeled folic acid derivatives often result in high and sustained renal uptake due to the high expression of folate receptor alpha and proton-coupled folate transporter in the proximal tubules of the kidney. ²³ It has been reported that radiolabeled folic acid derivatives have built-in albumin binders, which significantly prolong blood residence time, increase tumor uptake, and improve the uptake ratio of tumor to kidney. ²³

Recently, attempts have also been made to use this strategy to design PSMA-targeted endo-radiotherapeutic agents with albumin binding motifs. ^24-28 In the reported albumin-binding PSMA-targeted drugs ¹⁷⁷Lu-PSMa-aLB-02、¹⁷⁷ Lu-PSMA-alB-056 and ¹⁷⁷ Lu-RPS-063, the doses of radiation for tumors expressing PSMA were 1.8-fold, 2.3-fold and 3.8-fold higher than ¹⁷⁷ Lu-PSMA-617. ^26-28 Furthermore, the median survival prolongation of ¹⁷⁷Lu-PSMa-aLB-056.²⁷ mice treated with ¹⁷⁷ Lu-PSMA-617 or ¹⁷⁷ Lu-PSMA-aLB-056 compared to control mice treated with physiological saline was further assessed in a radiation therapy study of mice bearing PSMA-expressing PC-3PIP tumors. Most importantly, the use of only 2MBq ¹⁷⁷ Lu-PSMA-aLB-056 resulted in slightly better median survival (36 vs. 32 days) compared to ¹⁷⁷ Lu-PSMA-617 using 5 MBq.

In this example, the binding of a novel albumin binder was used to further enhance tumor uptake of ¹⁷⁷ Lu-PSMA-617, which is the most studied PSMA-targeted endo-radiation therapeutic. The most common albumin binding motif reported in the literature consists of D-Lys, which is acylated with the epsilon-amino group of 4- (p-iodophenyl) butanoic acid. ^22-23 Since the α -carboxyl group of D-Lys is part of the albumin binding motif, it cannot be bound to peptides by solid phase synthesis. ²⁹ As shown in the structure of Lu-HTK01169, a Glu residue was used instead of D-Lys. As a result, the carboxyl group on the Glu side chain can be used to bind albumin, and the α -carboxyl group can be bound to the peptide by solid phase synthesis. As shown in this example, modification of the linkage between DOTA chelator and PSMA-targeted Lys-urea-Glu did not adversely affect efficacy, confirming a report that such linkage modification was well tolerated. ¹⁷ Indeed, as shown in this example, a 6-fold improvement in PSMA binding was observed for Lu-HTK01169 compared to Lu-PSMA-617 (K _i value: 0.04.+ -. 0.01 vs. 0.24.+ -. 0.06 nM). Without wishing to be bound by theory, the improved PSMA binding may be due to the introduction of highly lipophilic 4- (p-iodophenyl) butanoyl.

The ability of ¹⁷⁷ Lu-HTK01169 to bind albumin was assessed by a plasma protein binding assay. Only <1% 177Lu-HTK01169 was observed under the same conditions compared to about 17% free ¹⁷⁷ Lu-PSMA-617, demonstrating the ability of the albumin binder modified derivatives to interact with plasma proteins.

The addition of albumin binders to extend blood residence time and maximize tumor uptake has been demonstrated by SPECT/CT and biodistribution studies. ¹⁷⁷ Lu-HTK01169 not only showed improved tumor uptake peaks (¹⁷⁷Lu-HTK 01169：55.9±12.5％ID/g;¹⁷⁷ Lu-PSMA-617:15.1.+ -. 5.58% ID/g), but most importantly, uptake was sustained, rather than decreasing over time as in ¹⁷⁷ Lu-PSMA-617. Without wishing to be bound by theory, this may be due in part to the increased PSMA binding of Lu-HTK01169 as compared to Lu-PSMA-617. The improved uptake and longer residence time provided an 8.3-fold higher radiation dose to the LNCaP tumor burden compared to ^177L u-PSMA-617. This design strategy may be more important for longer half-life radioisotopes, such as the alpha emitter ²²⁵Ac(t_1/2：²²⁵Ac,9.95d;¹⁷⁷ Lu,6.65 d). ²²⁵ Ac, currently in clinical use, was extracted from ²²⁹ Th and provided in limited supply. ^30-31 Conversion from ²²⁵ Ac-PSMA-617 to ²²⁵ Ac-HTK01169 may significantly increase the number of patients that can be treated with ²²⁵ Ac-labeled PSMA-targeted radioligand.

This example shows that the size of LNCaP tumor burden is rapidly reduced over time by injecting ¹⁷⁷ Lu-PSMA-617 or ¹⁷⁷ Lu-HTK 0169 of 37MBq (FIG. 2). The injected radiation dose of 37MBq for acquiring high resolution SPECT images may exceed the ¹⁷⁷ Lu-HTK01169 dose required to treat LNCaP tumors. Thus, the internal radiation therapy study in this example compared median survival of mice treated with either ¹⁷⁷ Lu-PSMA-617 or ¹⁷⁷ Lu-HTK01169 at 18.5MBq, and ¹⁷⁷ Lu-HTK01169 at only one half (9.3 MBq), one quarter (4.6 MBq) or one eighth (2.3 MBq). An eighth dose (2.3 MBq) of ¹⁷⁷ Lu-HTK01169 did not produce a similar median survival compared to ¹⁷⁷ Lu-HTK01169 (18.5 MBq, table 6) predicted by dosimetry data. However, median survival was observed to be slightly better for mice treated with a quarter dose (4.5 MBq) of ¹⁷⁷ Lu-HTK01169 than for mice treated with 18.5MBq of ¹⁷⁷ Lu-PSMA-617 (61 days versus 58 days, table 6).

Of the reported albumin binder-bound PSMA-targeted endo-radiotherapeutic agents, only ¹⁷⁷ Lu-PSMA-aLB-056 was evaluated in the radiotherapy study and compared directly with ¹⁷⁷ Lu-PSMA-617. ²⁷ The findings of this example differ from the findings reported by Umbricht et al for ¹⁷⁷ Lu-PSMA-aLB-056. ²⁷ For the tumor model, the unmodified endogenous prostate cancer cell line LNCaP was used in this example. Evaluation of ¹⁷⁷ Lu-PSMA-aLB-056 used PC-3PIP, a transduced cell line whose PSMA expression levels were much higher than LNCaP cells. ²⁷ Thus, the therapeutic doses (2 and 5 MBq) of ¹⁷⁷ Lu-PSMA-aLB-056 and ¹⁷⁷ Lu-PSMA-617 in the previously reported studies were lower than the doses (2.3-18.5 MBq) used in this example. the second difference is the size of the tumor. Unlike the 100mm ³ average tumor size used to evaluate ¹⁷⁷ Lu-PSMA-ALB-056, in this example, the tumor size ranges from 531-640mm ³ when starting treatment with ¹⁷⁷ Lu-PSMa-617 or ¹⁷⁷ Lu-HTK 01169. The larger tumor in this example may be more resistant to treatment, and then require a higher radiation dose to achieve similar growth inhibition.

Compared to ¹⁷⁷ Lu-PSMA-617, albumin binder-bound ¹⁷⁷ Lu-HTK01169 provided a 3.7-fold high peak uptake and 8.3-fold total dose of radiation to LNCaP tumor burden. Radiation therapy studies in mice bearing LNCaP tumors also showed that only a quarter dose of ¹⁷⁷ Lu-PSMA-617 activity was required to achieve a similar therapeutic effect for ¹⁷⁷ Lu-HTK 01169. HTK01169 radiolabeled with ¹⁷⁷ Lu or ²²⁵ Ac may also produce similar or improved radiotherapeutic effects when transformed into clinic, with only a partial dosing activity of ¹⁷⁷ Lu-PSMA-617. The newly introduced albumin binder in HTK01169 can be constructed directly on the solid phase along the peptide extension. Based on potentially valid data obtained from ¹⁷⁷ Lu-HTK01169, this new albumin binding motif might be applied to other (radioactive) peptides to extend their blood residence time and maximize therapeutic effect.

EXAMPLE 2 improved Metal chelating PSMA binding Compounds

2.1 Materials and methods

2.11 General procedure

All chemicals and solvents were commercial products and were used without further purification. PSMA-targeted peptides were synthesized using a solid phase method on AAPPTec (lewis wilt, kenta) Endeavor 90 peptide synthesizer. Purification and quality control of cold and radiolabeled peptides was performed on an Agilent HPLC system equipped with a type 1200 quaternary pump, a type 1200 UV absorbance detector (set at 220 nm) and a Bioscan (Washington, D.C.) sodium iodide scintillation detector. The operation of the Agilent HPLC system was controlled by Agilent ChemStation software. The HPLC columns used were semi-preparative (Luna C18, 5. Mu.250X 10 mm) and analytical (Luna C18, 5. Mu.250X 4.6 mm) columns, available from Phenomenex (Tolans, calif.). The collected HPLC eluate containing the desired peptide was lyophilized using a Labconco (kansase city, miso) FreeZone 4.5.5 Plus freeze dryer. Mass analysis was performed using an AB SCIEX (framingham, ma) 4000QTRAP mass spectrometer system with ESI ion source. C18 Sep-Pak cartridge (1 cm ³, 50 mg) was obtained from Waters (Miphora isthmus, massachusetts). ⁶⁸ Ga was eluted from iThemba laboratory (samercase west, south africa) generators and purified using a DGA resin column of EichromTechnologies LLC (rassal, il). ⁶⁸ Radioactivity of Ga-labeled peptide Capntec (New Jersey lamda)Dose calibrator was measured and radioactivity of mouse tissue collected from biodistribution studies was counted using PERKIN ELMER (waltherm, ma) Wizard2 2480 automatic gamma counter.

2.12 Synthesis of HTK 03026, HTK03027, HTK03029 and HTK03041

The structures of HTK03026, HTK03027, HTK03029 and HTK03041 are shown below:

Solid phase synthesis of HTK3026, HTK03027, HTK03029 and HTK03041 was modified according to the literature. ¹⁶ Fmoc-Lys (ivDde) -Wang resin (0.3 mmol,0.61mmol/g load) was suspended in DMF for 30 min. Fmoc was then removed by treating the resin with 20% piperidine in DMF (3X 8 min). The isocyanate derivative of di-tert-butyl glutamate (3 eq) was prepared according to literature procedure ¹⁷ and added to the lysine fixed resin and reacted for 16 hours. After washing the resin with DMF, the ivDde-protecting group was removed with 2% hydrazine in DMF (5X 5 min). Fmoc-2-Aoc-OH (for HTK 03026), fmoc-Ala (2-Anth) -OH (for HTK 03027), fmoc-Ala (1-pyridinyl) -OH (for HTK 03029) or Fmoc-Ala (9-Anth) -OH (for HTK 03041) was coupled to the side chain of Lys using Fmoc-protected amino acids (3 eq), HBTU (3 eq), HOBT (3 eq) and N, N-diisopropylethylamine (8 eq). Extension was then continued by adding Fmoc-tranexamic acid and finally DOTA-tris (t-bu) ester (2- (4, 7, 10-tris (2- (t-butoxy) -2-oxoalkyl) -1,4,7, 10) -tetraazacyclodode-1-yl) acetic acid.

The peptide was then deprotected and cleaved from the resin by treatment with 95/5 trifluoroacetic acid (TFA)/Triisopropylsilane (TIS) for 2 hours at room temperature. After filtration, the peptide was precipitated by adding cold diethyl ether to the TFA solution. The crude peptide was purified by HPLC using a semi-preparative column. The eluate containing the desired peptide is collected, pooled and lyophilized. For HTK03026, HPLC conditions were 27% acetonitrile in water, 0.1% TFA, flow rate 4.5 mL/min. The residence time was 10.7 minutes. ESI-MS calculated [ M+H ] ⁺ 986.5 for HTK 03026C ₄₅H₇₅N₉O₁₆, found [ M+H ] ⁺ 986.6. For HTK03027, HPLC conditions were 32% acetonitrile in water, 0.1% TFA, flow rate 4.5 mL/min. The residence time was 7.1 minutes. ESI-MS calculated [ M+H ] ⁺ 1092.5 for HTK 03027C ₅₃H₇₄N₉O₁₆, found [ M+H ] ⁺ 1094.6. For HTK03029, HPLC conditions were 33% acetonitrile in water, 0.1% TFA, and flow rate was 4.5 mL/min. The residence time was 7.3 minutes. ESI-MS calculated [ M+H ] ⁺ 1116.5 for HTK 03029C ₅₅H₇₄N₉O₁₆, found [ M+H ] ⁺ 1116.6. For HTK03041, HPLC conditions were 31% acetonitrile in water, 0.1% TFA, flow rate 4.5 mL/min. The residence time was 7.2 minutes. ESI-MS: HTK 03041C ₅₃H₇₄N₉O₁₆ d found [ M+H ] ⁺ 1092.5 [ M+H ] ⁺ 1092.6.

2.13 The combination of HTK03024, HTK03055, HTK03056, HTK03058, HTK03082, HTK03085, HTK03086, HTK03087, HTK03089 and HTK03090

The structures of HTK03024, HTK03055, HTK03056, HTK03058, HTK03085, HTK03086, HTK03087, HTK03089 and HTK03090 are as follows:

R＝I(HTK 03024)、CI(HTK 03055)、H(HTK03056)、Br(HTK03058)、F(HTK03085)、OCH₃(HTK03086)、NH₂(HTK03087)、NO₂(HTK03089) or CH ₃ (HTK 03090).

The structure of HTK03082 is shown below:

Fmoc-Lys (ivDde) -Wang resin (0.3 mmol,0.61mmol/g load) was suspended in DMF for 30 min. Fmoc was then removed by treating the resin with 20% piperidine in DMF (3X 8 min). The isocyanate derivative of di-tert-butyl glutamate (3 eq) was prepared according to literature procedure ¹⁷ and added to the lysine fixed resin and reacted for 16 hours. After washing the resin with DMF, the ivDde-protecting group was removed with 2% hydrazine in DMF (5X 5 min). Fmoc-2-Nal-OH was then coupled to the side chain of Lys by solid phase peptide synthesis using Fmoc-based chemistry, followed by Fmoc-tranexamic acid, fmoc-Lys (ivDde) -OH and Fmoc-Gly-OH. All couplings were performed in DMF using Fmoc protected amino acids (3 eq), HBTU (3 eq), HOBT (3 eq), and DIEA (8 eq). Then, continuing to extend, 4- (p-iodophenyl) butanoic acid (for HTK 03024), 4- (p-chlorophenyl) butanoic acid (for HTK 03055), 4-phenylbutanoic acid (for HTK 03056), 4- (p-bromophenyl) butanoic acid (for HTK 03058), 3-phenylpropionic acid (for HTK 03082), 4- (p-fluorophenyl) butanoic acid (for HTK 03085), 4- (p-methoxyphenyl) butanoic acid (for HTK 03086), 4- (p-tert-butoxycarbonyl) aminophenyl) butanoic acid (for HTK 03087), 4- (p-nitrophenyl) butanoic acid (for HTK 03089) or 4- (p-tolyl) butanoic acid (for HTK 03090) are coupled to the same peptide using Fmoc-based chemistry to bind the resin. After selective removal of the ivDde protecting group with 2% hydrazine in DMF (5 x 5 min), the chelator DOTA was then coupled to the side chain of Lys to give the precursor.

The peptide was then deprotected and cleaved from the resin by treatment with 95/5 trifluoroacetic acid (TFA)/Triisopropylsilane (TIS) for 2 hours at room temperature. After filtration, the peptide was precipitated by adding cold diethyl ether to the TFA solution. The crude peptide was purified by HPLC using a semi-preparative column. The eluate containing the desired peptide is collected, pooled and lyophilized. For HTK03024, HPLC conditions were 37% acetonitrile in water, 0.1% TFA, flow rate 4.5 mL/min. The residence time was 8.8 minutes. ESI-MS calculated for HTK 03024C ₆₇H₉₆N₁₂O₁₉ I [ M+H ] ⁺ 1499.6, found [ M+H ] ⁺ 1499.6. For HTK03055, HPLC conditions were 35% acetonitrile in water, 0.1% TFA, flow rate 4.5 mL/min. The residence time was 9.7 minutes. ESI-MS calculated for HTK 03055C ₆₇H₉₆N₁₂O₁₉ Cl [ M+H ] ⁺ 1407.7, found [ M+H ] ⁺ 1407.7. For HTK03056, HPLC conditions were 0-80% acetonitrile in water, 0.1% TFA, flow rate 4.5 mL/min, 20 min. The residence time was 13.4 minutes. ESI-MS calculated [ M+H ] ⁺ 1373.7 for HTK 03056C ₆₇H₉₇N₁₂O₁₉, found [ M+H ] ⁺ 1373.8. For HTK03058, HPLC conditions are 0-80% acetonitrile in water, 0.1% TFA, flow rate 4.5 mL/min, 20 min. The residence time was 13.4 minutes. ESI-MS calculated [ M+H ] ⁺ 1451.6 for HTK 03058C ₆₇H₉₆N₁₂O₁₉ Br, found [ M+H ] ⁺ 1451.6. For HTK03082, HPLC conditions were 31% acetonitrile in water, 0.1% TFA, and flow rate was 4.5 mL/min. The residence time was 11.1 minutes. ESI-MS calculated 1359.7 for HTK 03082C ₆₆H₉₅N₁₂O₁₉, found [ M+H ] ⁺ 1359.9. For HTK03085, HPLC conditions were 34% acetonitrile in water, 0.1% TFA, and flow rate was 4.5 mL/min. The residence time was 9.0 minutes. ESI-MS calculated for HTK 03085C ₆₇H₉₆N₁₂O₁₉ F [ M+H ] ⁺ 1391.7, found [ M+H ] ⁺ 1391.9. For HTK03086, HPLC conditions are 33% acetonitrile in water, 0.1% TFA, flow rate of 4.5 mL/min. The residence time was 9.1 minutes. ESI-MS calculated [ M+H ] ⁺ 1403.7 for HTK 03090C ₆₈H₉₉N₁₂O₂₀, found [ M+H ] ⁺ 1404.1.1404. For HTK03087, HPLC conditions were 23% acetonitrile in water, 0.1% TFA, flow rate 4.5 mL/min. The residence time was 13.9 minutes. ESI-MS calculated ⁺ 1388.7 for HTK 03087C ₆₇H₉₈N₁₃O₁₉, found [ M+H ]1389.0. For HTK03089, HPLC conditions are 33% acetonitrile in water, 0.1% TFA, flow rate of 4.5 mL/min. The residence time was 10.6 minutes. ESI-MS calculated [ M+H ] ⁺ 1418.7 of HTK 03089C ₆₇H₉₆N₁₃O₂₁, found [ M+H ] ⁺ 1419.0. For HTK03090, HPLC conditions were 35% acetonitrile in water, 0.1% TFA, flow rate 4.5 mL/min. The residence time was 9.1 minutes. ESI-MS calculated [ M+H ] ⁺ 1387.7 for HTK 03090C ₆₈H₉₉N₁₂O₁₉, found [ M+H ] ⁺ 1387.9.

2.14 Synthesis of Ga-labeled standards

To prepare Ga-labeled standards, solutions of each precursor were incubated with GaCl ₃ (5 eq.) in NaOAc buffer (0.1 m,500 μl, pH 4.2) at 80 ℃ for 15 minutes. The reaction mixture was then purified by HPLC using a semi-preparative column and the HPLC eluent containing the desired peptide was collected, pooled and lyophilized. For Ga-HTK03026, HPLC conditions are 27% acetonitrile in water, 0.1% TFA, flow rate of 4.5 mL/min. The residence time was 9.4 minutes. ESI-MS calculated for Ga-HTK 03026C ₄₄H₇₃N₉O₁₆ Ga [ M+H ] ⁺ 1052.4, found [ M+H ] ⁺ 1052.5. For Ga-HTK03027, the HPLC conditions were 32% acetonitrile in water, 0.1% TFA, flow rate was 4.5 mL/min. The residence time was 9.5 minutes. ESI-MS calculated for Ga-HTK 03027C ₅₃H₇₂N₉O₁₆ Ga [ M+H ] ⁺ 1159.4, found [ M+H ] ⁺ 1161.4. For HTK03029, HPLC conditions were 33% acetonitrile in water, 0.1% TFA, and flow rate was 4.5 mL/min. The residence time was 10.3 minutes. ESI-MS, calculated for Ga-HTK 03029C ₅₅H₇₂N₉O₁₆ Ga [ M+H ] ⁺ 1183.4, found [ M+H ] ⁺ 1183.4. For Ga-HTK03041, the HPLC conditions are 31% acetonitrile in water, 0.1% TFA, flow rate 4.5 mL/min. The residence time was 9.3 minutes. ESI-MS calculated for Ga-HTK 03041C ₅₃H₇₂N₉O₁₆ Ga [ M+H ] ⁺ 1159.4, found [ M+H ] ⁺ 1159.4. For Ga-HTK03024, the HPLC conditions are 39% acetonitrile in water, 0.1% TFA, flow rate 4.5 mL/min. The residence time was 8.0 minutes. ESI-MS calculated [ M+H ] ⁺ 1565.5 for Ga-HTK 03024C ₆₇H₉₃N₁₂O₁₉ IGa, found [ M+H ] ⁺ 1565.5. For Ga-HTK03055, the HPLC conditions are 35% acetonitrile in water, 0.1% TFA, flow rate 4.5 mL/min. the residence time was 12.7 minutes. ESI-MS calculated [ M+H ] ⁺ 1474.6 for Ga-HTK 03055C ₆₇H₉₄N₁₂O₁₉ ClGa, found [ M+H ] ²⁺ 738.4. For Ga-HTK03056, HPLC conditions are 34% acetonitrile in water, 0.1% TFA, flow rate of 4.5 mL/min. The residence time was 9.0 minutes. ESI-MS, ga-HTK 03056C ₆₇H₉₄N₁₂O₁₉ Ga calculated [ M+H ] ⁺ 1439.6, found [ M+H ] ⁺ 1439.8. For Ga-HTK03058, HPLC conditions are 34% acetonitrile in water, 0.1% TFA, flow rate of 4.5 mL/min. The residence time was 10.3 minutes. ESI-MS calculated [ M+H ] ⁺ 1517.5 of Ga-HTK 03058C ₆₇H₉₃N₁₂O₁₉ BrGa, found [ M+H ] ⁺ 1518.0. For Ga-HTK03082, HPLC conditions are 31% acetonitrile in water, 0.1% TFA, flow rate of 4.5 mL/min. The residence time was 12.5 minutes. ESI-MS, calculated for Ga-HTK03082C ₆₆H₉₃N₁₂O₁₉ Ga [ M+H ] ⁺ 1426.6, found [ M+H ] ⁺ 1426.9. For Ga-HTK03085, HPLC conditions are 34% acetonitrile in water, 0.1% TFA, flow rate of 4.5 mL/min. The residence time was 9.0 minutes. ESI-MS, calculated [ M+H ] ⁺ 1458.6 of Ga-HTK 03085C ₆₇H₉₄N₁₂O₁₉ FGa, found [ M+H ] ⁺ 1459.6. For Ga-HTK03086, HPLC conditions are 33% acetonitrile in water, 0.1% TFA, flow rate of 4.5 mL/min. The residence time was 10.7 minutes. ESI-MS calculated for Ga-HTK 03086C ₆₈H₉₆N₁₂O₂₀ Ga 1469.6, found [ M+H ] ⁺ 1469.8. For Ga-HTK03087, HPLC conditions are 23% acetonitrile in water, 0.1% TFA, flow rate of 4.5 mL/min. The residence time was 14.7 minutes. ESI-MS calculated for Ga-HTK 03087C ₆₇H₉₆N₁₃O₁₉ Ga [ M+H ] ⁺ 1455.6, found [ M+H ] ⁺ 1455.8. For Ga-HTK03089, HPLC conditions are 33% acetonitrile in water, 0.1% TFA, flow rate of 4.5 mL/min. The residence time was 12.0 minutes. ESI-MS, calculated [ M+H ] ⁺ 1485.6 of Ga-HTK 03089C ₆₇H₉₄N₁₃O₂₁ Ga, found [ M+H ] ⁺ 1485.9. For Ga-HTK03090, the HPLC conditions are 35% acetonitrile in water, 0.1% TFA, flow rate 4.5 mL/min. The residence time was 11.3 minutes. ESI-MS calculated for Ga-HTK 03090C ₆₈H₉₇N₁₂O₁₉ Ga [ M+H ] ⁺ 1454.6, found [ M+H ] ⁺ 1455.8.

2.15 Cell culture

LNCaP cell lines were obtained from ATCC (LNCap clone FGC, CRL-1740). It is established from the metastatic site of the lymph node on the left collarbone of human prostate cancer. Cells were cultured in PRMI 1640 medium supplemented with 10% FBS, penicillin (100U/mL) and streptomycin (100. Mu.g/mL) at 37℃and 5% CO ₂ in a humidified incubator. Cells grown to 80-90% confluence were then washed with sterile phosphate buffered saline (1 XPBS pH 7.4) and trypsinized. The number of cells collected was counted using a Hausser Scientific (Huo Shem pa) cytometer.

2.16 Synthesis of ⁶⁸ Ga-labeled Compound

⁶⁸ Ga purified in 0.5mL of water was added to a 4mL glass bottle pre-filled with 0.7mLHEPES buffer (2M, pH 5.0) and 50. Mu.g DOTA precursor. The radiolabelling reaction was carried out under microwave heating for 1 minute. The reaction mixtures were purified by HPLC using the same semi-preparative column and conditions provided in section 2.14 to purify their respective nonradioactive Ga-labeled standard solutions.

2.17 PET/CT imaging and biodistribution

Imaging and biodistribution experiments were performed using nodscd 1L2rγko male mice. Mice were anesthetized with 2% isoflurane in oxygen and subcutaneously implanted with 1×10 ⁷ LNCaP cells behind the left shoulder. Mice were imaged or used for biodistribution studies when tumors grew to a diameter of 5-8mm within 5-6 weeks.

PET imaging experiments were performed using Siemens Inveon microPET/CT scanner. Each tumor-bearing mouse was injected under anesthesia with 6-8MBq of 68 Ga-labeled tracer (2% isoflurane in oxygen) via the tail vein. The mice were allowed to resume consciousness and were free to move around in the cages. After 50 minutes, the mice were again allowed to inhale 2% isoflurane in oxygen, sedated, and placed in a scanner. First a CT scan is performed for 10 minutes, and positioning and attenuation correction are performed after segmentation to reconstruct PET images. Static PET imaging was then performed for 10 minutes to determine uptake of tumors and other organs. During the collection, the mice are kept warm by the heating pad. For imaging studies obtained 3 hours after injection (intraperitoneal injection), mice were placed in a micro PET/CT scanner 170 minutes after intraperitoneal injection. CT acquisitions were then performed as described above, with 15 minutes of static PET imaging to determine uptake in tumors and other organs.

For biodistribution studies, mice were injected with radiotracer as described above. At predetermined time points (1 or 3 h), mice were inhaled 2% isoflurane, anesthetized, and euthanized by inhalation of CO ₂. Blood is immediately drawn from the heart and the target organ/tissue is collected. The collected organs/tissues were weighed and counted using an automatic gamma counter. The uptake per organ/tissue was normalized to the injected dose using a standard curve and expressed as a percentage of injected dose per gram of tissue (% ID/g).

2.2 Results

The results of this example are shown in tables 7-10 and FIGS. 13-16. In combination with the results of example 1, these results indicate that the various compounds comprised in formulas 1-a and 1-b would be particularly useful.

TABLE 7 biological distribution data and tumor to background contrast in ⁶⁸ Ga-tagged HTK03026, HTK03027, HTK03029 and HTK03041 in LNCaP tumor-loaded mice bearing PSMA-expressing cells.

TABLE 8 biological distribution data and tumor to background contrast for ⁶⁸ Ga-labeled HTK03089 and HTK03090 in LNCAP tumor-loaded mice bearing PSMA.

U.S. provisional application No. 62/575,460 filed on 10/22/2017 is incorporated herein by reference in its entirety. To the extent that there may be a divergence between the definitions provided in the present application and the definitions provided in the files loaded by reference, the definitions in the present application should take precedence over the definitions in the files loaded by reference.

One or more embodiments are described herein. It will be apparent, however, to one skilled in the art that many changes and modifications can be made without departing from the scope of the application as defined in the following claims.

Reference to the literature

1.Carter,R.E.;Feldman,A.R.;Coyle,J.T.Prostate-specific membrane antigen is a hydrolase with substrate and pharmacologic characteristics of a neuropeptidase.Proc.Natl.Acad.Sci.U.S.A.1996,93,749-753.

2.Silver,D.A.;Pellicer,I.;Fair,W.R.;Heston,W.D.;Cordon-Cardo,C.Prostate-specific membrane antigen expression in normal and malignant human tissues.Clin.Cancer Res.1997,3,81-85.

3.Sokoloff,R.L.;Norton,K.C.;Gasior,C.L.;Marker,K.M.;Grauer,L.S.A dual-monoclonal sandwich assay for prostate-specific membrane antigen：levels in tissues,seminal fluid and urine.Prostate 2000.43.150-157.

4.Bander,N.H.;Milowsky,M.I.;Nanus,D.M.;Kostakoglu,L.;Vallabhajosula,S.;Goldsmith,S.J.Phase I trial of ¹⁷⁷lutetium-labeled J591,a monoclonal antibody to prostate-specific membrane antigen,in patients with androgen-independent prostate cancer.J.Clin.Oncol.2005,23,4591-4601.

5.Afshar-Oromieh,A.;Haberkorn,U.;Zechmann,C.;Armor,T.;Mier,W.;Spohn,F.;Debus,N.;Holland-Letz,T.;Babich,J.;Kratochwil,C.Repeated PSMA-targeting radioligand therapy of metastatic prostate cancer with ¹³¹I-MIP-1095.Eur.J.Nucl.Med.Mol.Imaging 2017,44,950-959.

6.Heck,M.M.;Retz,M.;D'Alessandria,C.;Rauscher,I.;Scheidhauer,K.;Maurer,T.;Storz,E.;Janssen,F.;Schottelius,M.;Wester,H.J.;Gschwend,J.E.;Schwaiger,M.;Tauber,R.;Eiber,M.Systemic radioligand therapy with ¹⁷⁷Lu labeled prostate specific membrane antigen ligand for imaging and therapy in patients with metastatic castration resistant prostate cancer.J.Urol.2016,196.382-391.

7.Kratochwil,C.;Giesel,F.L.;Stefanova,M.;Benesova,M.;Bromzel,M.;Afshar-Oromieh,A.;Mier,W.;Eder,M.;Kopka,K.;Haberkorn,U.PSMA-targeted radionuclide therapy of metastatic castration-resistant prostate cancer with ¹⁷⁷Lu-labeled PSMA-617.J.Nucl.Med.2016,57,1170-1176.

8.Rahbar,K.;Bode,A.;Weckesser,M.;Avramovic,N.;Claesener,M.;Stegger,L.;Bogemann,M.Radioligand thcrapy with ¹⁷⁷Lu-PSMA-617 as a novel therapeutic option in patients with metastatic castration resistant prostate cancer.Clin.Nucl.Med.2016.41.522-528.

9.Fendler,W.P.;Reinhardt,S.;Ilhan,H.;Delker,A.;Boning,G.;Gildehaus,F.J.;Stief,C.;Bartenstein,P.;Gratzke,C.;Lehner,S.;Rominger,A.Preliminary experience with dosimetry,response and patient reported outcome after ¹⁷⁷Lu-PSMA-617 therapyfor metastatic castration-resistant prostate cancer.Oncotarget 2017,8,3581-3590.

10.Rahbar,K.;Ahmadzadehfar,H.;Kratochwil,C.;Haberkorn,U.;Schafers,M.;Essler,M.;Baum,R.P.;Kulkarni,H.R.;Schmidt,M.;Drzezga,A.;Bartenstein,P.;Pfestroff,A.;Luster,M.;Lutzen,U.;Marx,M.;Prasad,V.;Brenner,W.;Heinzel,A.;Mottaghy,F.M.;Ruf,J.;Meyer,P.T.;Heuschkel,M.;Eveslage,M.;Bogemann,M.;Fendler,W.P.;Krause,B.J.German multicenter study investigating ¹⁷⁷Lu-PSMA-617 radioligand therapy in advanced prostate cancer patients.J.Nucl.Med.2017,58,85-90.

11.Ahmadzadehfar,H.;Wegen,S.;Yordanova,A.;Fimmers,R.;Kurpig,S.;Eppard,E.;Wei,X.;Schlenkhoff,C.;Hauser,S.;Essler,M.Overall survival and response pattern of castration-resistant metastatic prostate cancer to multiple cycles of radioligand therapy using[¹⁷⁷Lu]Lu-PSMA-617.Eur.J.Nucl.Med.Mol.Imaging 2017,44,1448-1454.

12.Brauer,A.;Grubert,L.S.;Roll,W.;Schrader,A.J.;Schafers,M.;Bogemann,M.;Rahbar,K.¹⁷⁷Lu-PSMA-617 radioligand therapy and outcome in patients with metastasized castration-resistant prostate cancer.Eur.J.Nucl.Med.Mol.Imaging 2017,44,1663-1670.

13.Yadav,M.P.;Ballal,S.;Tripathi,M.;Damle,N.A.;Sahoo,R.K.;Seth,A.;Bal,C.¹⁷⁷Lu-DKFZ-PSMA-617 therapy in metastatic castration resistant prostate cancer：safety,efficacy,and quality of life assessment.Eur.J.Nucl.Med.Mol.Imaging 2017,44,81-91.

14.Hofman,M.S.;Violet,J.;Hicks,R.J.;Ferdinandus,J.;Thang,S.P.;Akhurst,T.;Iravani,A.;Kong,G.;Kumar,A.R.;Murphy,D.G.;Eu,P.;Jackson,P.;Scalzo,M.;Williams,S.G.;Sandhu,S.[¹⁷⁷Lu]-PSMA-617 radionuclide treatment in patients with metastatic castration-resistant prostate cancer(LuPSMA trial)：a single-centte,single-arm,phase 2 study.Lancet Oncol.2018,19,825-833.

15.Kratochwil,C.;Bruchertseifer,F.;Giesel,F.L.;Weis,M.;Verburg,F.A.;Mottaghy,F.;Kopka,K.;Apostolidis,C.;Haberkorn,U.;Morgenstern,A.²²⁵Ac-PSMA-617 for PSMA-targeted α-radiation therapy of metastatic castration-resistant prostate cancer.J.Nucl.Med.2016,57,1941-1944.

16.Benesova,M.;Schafer,M.;Bauder-Wust,U.;Afshar-Oromieh,A.;Kratochwil,C.;Mier,W.;Haberkorn,U.;Kopka,K.;Eder,M.Preclinical evaluation of a tailor-made DOTA-conjugated PSMA inhibitor with optimized linker moiety for imaging and endoradiotherapy of prostate cancer.J.Nucl.Med.2015,56,914-920.

17.Benesova,M.;Bauder-Wust,U.;Schafer,M.;Klika,K.D.;Mier,W.;Haberkorn,U.;Kopka,K.;Eder,M.Linker modification strategies to control the prostate-specific membrane antigen(PSMA)-targeting and pharmacokinetic properties of DOTA-conjugated PSMA inhibitors.J.Med.Chem.2016,59,1761-1775.

18.Kuo,H.T.;Pan,J.;Zhang,Z.;Lau,J.;Merkens,H.;Zhang,C.;Colpo,N.;Lin,K.S.;Benard,F.Effects of linker modification on tumor-to-kidney contrast of ⁶⁸Ga-labeled PSMA-targeted imaging probes.Mol.Pharmaceutics 2018,doi：10.1021/acs.molpharmaceut.8b00499

19.Meckel,M.;Kubicek,V.;Hermann,P.;Miederer,M.;Rosch,F.A DOTA based bisphosphonate with an albumin binding moiety for delayed body clearance for bone targeting.Nucl.Med.Biol.2016,43,670-678.

20.Harada,N.;Kimura,H.;Onoe,S.;Watanabe,H.;Matsuoka,D.;Arimitsu,K.;Ono,M,;Saji,H.Synthesis and biological evaluation of novel ¹⁸F-labeled probes targeting prostate-specific membrane antigen for positron emission tomography of prostate cancer.J.Nucl.Med.2016,57,1978-1984.

21.Chatalic,K.L.S.;Heskamp,S.;Konijnenberg,M.;Molkenboer-Kuenen,J.D.M.;Franssen,G.M.;Clahsen-van Groningen,M.C.;Schottelius,M.;Wester,H.J.;van Weerden,W.M.;Boerman,O.C.;de Jong,M.Towards personalized treatment of prostate cancer：PSMA I&T,a promising prostate-specific membrane antigen-targeted theranostic agent.Theranostics 2016,6,849-861.

22.Dumelin,C.E.;Trussel,S.;Buller,F.;Trachsel,E.;Bootz,F.;Zhang,Y.;Mannocci,L.;Beck,S.C.;Drumea-Mirancea,M.;Seeliger,M.W.;Baltes,C.;Muggler,T.;Kranz,F.;Rudin,M.;Melkko,S.;Scheuermann,J.;Neri,D.A portable albumin binder from a DNA-encoded chemical library.Angew.Chem.Int.Ed.2008,47,3196-3201.

23.Muller,C.;Struthers,H.;Winiger,C.;zhernosekov,K.;Schibli,R.DOTA conjugate with an albumin-binding entity enables the first folic acid-targeted ¹⁷⁷Lu-radionuclide rumor therapy in mice.J.Nucl.Med.2013,54,124-131.

24.Choy,C.J.;Ling,X.;Geruntho,J.J.;Beyer,S.K.;Latoche,J.D.;Langton-Webster,B.;Anderson,C.J.;Berkman,C.E.¹⁷⁷Lu-Labeled phosphoramidate-based PSMA inhibitors：The effect of an albumin binder on biodistribution and therapeutic efficacy in prostate tumor-bearing mice.Theranostics 2017.7.1928-1939.

25.Kelly,J.M.;Amor-Coarasa,A.;Nikolopoulou,A.;Wustemann,T.;Barelli,P.;Kim,D.;Williams,C.;Zheng,X.;Bi,C.;Hu,B.;Warren,J.D.;Hage,D.S.;DiMagno,S.G.;Babich,J.W.Dual-target binding ligands with modulated pharmacokinetics for endoradiotherapy of prostate cancer.J.Nucl.Med.2017,58,1442-1449.

26.Benesova,M.;Umbricht,C.A.;Schibli,R.;Müller,C.Albumin-binding PSMA ligands：optimization ofthe tissue distribution profile.Mol.Pharmaceutics 2018,15,934-946.

27.Umbricht,C.A.;Benesova,M.;Schibli,R.;Müller,C.Preclinical development of novel PSMA-targeting radioligands：Modulation of albumin-binding properties to improve prostate cancer therapy.Mol.Pharmaceutics 2018,15,2297-2306.

28.Kelly,J.;Amor-Coarasa,A.;Ponnala,S.;Nikolopoulou,A.;Williams,C.;Schlyer,D.;Zhao,Y.;Kim,D.;Babich,J.W.Trifunctional PSMA-targeting constructs for prostate cancer with unprecedented localization to LNCaP tumors.Eur.J.Nucl.Med.Mol.Imaging 2018,doi：10.1007/s00259-018-4004-5.

29.Liu,z.;Chen,x.Simple Bioconjugate chemistry serves great clinical advances：albumin as a versatile platform for diagnosis and precision therapy.Chem.Soc.Rev.2016,45,1432-1456.

30.Apostolidis,C.;Molinet,R.;Rasmussen,G.;Morgenstern,A.Production of Ac-225 from Th-229 for targeted α therapy.Anal.Chem.2005,77,6288-6291.

31.Walsh,K.M.Brookhaven National Laboratory：Radioisotopes for medical imaging and disease treatment.J.Nucl.Med.2017.58,11N-12N.

37.Stabin MG,Sparks RB,Crowe E.OLINDA/EXM：the second-generation personal computer software for internal dose assessment in nuclear medicine.J Nucl Med.2005;46：1023-1027.

38.Keenan MA,Stabin MG,Segars WP,Fernald MJ.RADAR realistic animal model series for dose assessment.J Nucl Med.2010;51：471-476.

39.Stabin MG,Xu XG,Emmons MA,Segars WP,Shi C,Fernald MJ.RADAR reference adult,pediatric,and pregnant female phantom series for internal and external dosimetry.J Nucl Med.2012;53：1807-1813.

40.Stabin MG,Konijnenberg MW.Re-evaluation of absorbed fractions for photohs and electrons in spheres of various sizes.J Nucl Med.2000;41：149-160.

41.Kirschner AS,Ice RD,Beierwaltes WH.Radiation dosimetry of ¹³¹I-19-iodocholesterol：the pitfalls of using tissue concentration data.The author's reply.J Nucl Med.1975;16：248-249.

42.Wessels BW,Bolch WE,Bouchet LG,et al.Bone marrow dosimetry using blood-based models for radiolabeled antibody therapy：a multiinstitutional comparison.J Nucl Med.2004;45(10)：1725-1733.

Claims (11)

1. A compound having the structure:

Wherein X is absent.

2. A compound having the structure:

wherein X is ⁹⁰Y,⁶⁷Ga,⁶⁸Ga,¹⁷⁷Lu,²²⁵ Ac, or ¹¹¹ In.

3. The compound of claim 2, wherein X is ¹⁷⁷ Lu.

4. A compound having the structure:

Wherein R is

5. A compound according to claim 4, wherein R is

6. A compound having the structure:

Wherein R is And wherein the compound binds to a radioactive metal.

7. A compound according to claim 6, wherein R is

8. A compound according to claim 6 or 7, wherein the radioactive metal is ⁶⁴Cu,¹¹¹ln,⁸⁹Zr,⁴⁴Sc,⁶⁸Ga,^99mTc,⁸⁶Y,¹⁵² Tb or ¹⁵⁵ Tb.

9. A compound according to claim 6 or 7, wherein the radioactive metal is ⁶⁸ Ga.

10. Use of a composition comprising a compound according to any one of claims 2 and 6-9 and a pharmaceutically acceptable excipient in the manufacture of a medicament for imaging a cancer expressing Prostate Specific Membrane Antigen (PSMA).

11. Use of a composition comprising a compound of claim 3 and a pharmaceutically acceptable excipient in the manufacture of a medicament for the treatment of prostate cancer.